ES2922081T3 - Virus-free cell lines and methods for obtaining them - Google Patents

Abstract

Current teachings are directed to novel virus-free cell lines derived from virus-contaminated target material, such as an organism or a cell line. Methods for obtaining virus-free cell lines obtained from virus-contaminated starting material are also provided. Exemplary virus-free cell lines include: novel cell lines derived from a Spodoptera Frugiperda cell line contaminated with SF-Rhabdovirus, wherein the novel cell lines lack SF-Rhabdovirus; and novel cell lines derived from a Trichoplusia Ni cell line contaminated with an alphanodavirus, wherein the novel cell line lacks an alphanodavirus. (Automatic translation with Google Translate, without legal value)

Description

DESCRIPTION

Virus-free cell lines and methods for obtaining them

Countryside

Current teachings generally refer to continuous Spodoptera frugiperda cell lines that are free of contaminating Sf-rhabdovirus. Current teachings also relate to methods for obtaining Sf-rhabdovirus-free Spodoptera frugiperda cell lines that are derived from cells or organisms that are contaminated with Sf-rhabdovirus.

Background

In vitro propagated cells can be broadly classified as primary cells or continuous cell lines, also called established cell lines. Primary cells can be obtained by isolating an organ or tissue from an organism and disaggregating it to create a mixture of individual cells. When primary cells are propagated in culture, they divide only a limited number of times before they lose their ability to proliferate, a genetically determined event known as senescence. However, some cells go through a process called transformation and gain the ability to divide indefinitely. These cells are called transformed cells or continuous cells. Compared to natural cells found in the tissue or organ from which they are derived, continuous cell lines often have genetic abnormalities, such as aneuploidy or heteroploidy, and lack the contact inhibition and anchorage dependence often seen in the primary cells.

Over the years, cultured cells used for bioproduction have repeatedly been found to be contaminated with viruses. For example, in the early 1960s it was discovered that adenovirus vaccines and poliovirus vaccines that were produced in primary Rhesus monkey kidney (RMK) cells were contaminated with simian virus 40 (SV40). SV40 was later shown to cause tumors in hamsters, and antibodies to SV40 were detected in people who had received inactivated poliovirus vaccine produced in primary RMK cells. In the 1970s, several lots of live measles, mumps, rubella, and polio vaccines were found to be contaminated with bacterial viruses known as bacteriophages. Avian leukosis virus (ALV) and endogenous avian virus (AEV) were found in attenuated yellow fever, measles, and mumps vaccines produced in chick embryo fibroblasts. The source of vaccine-associated ALVs and AEVs was thought to be endogenous retroviruses integrated into the chicken genome. More recently, several lots of rotavirus vaccines were found to be contaminated with infectious porcine circovirus-1 (PCV-1).

Since it was first described in the peer-reviewed literature in the early 1980s, the baculovirus-insect cell system (BICS) has become a widely recognized and widely used recombinant protein production platform. Advantages of BICS include its flexibility, speed, simplicity, eukaryotic protein processing capabilities, and ability to produce multi-subunit protein complexes. For nearly 30 years, BICS was used primarily to produce recombinant proteins for basic research in academic and industrial laboratories. More recently, however, BICS emerged as a true commercial manufacturing platform, now used to produce several biological products licensed for use in human medicine (CeRvARIX®, PROVENGE®, GLYBERA®, and FLUBLOK®) or veterinary ( PORCILIS® PESTI, BAYOVAC LCR E2®, CIRCUNVENT® PCV, INGELVAC CIRCOFLEX® and PORCILIS® PCV). In addition, BICS is being used to produce a variety of other biologics, including vaccine candidates against norovirus, parvovirus, Ebola virus, respiratory syncytial virus, and hepatitis E virus in various stages of human clinical trials.

The insect cell lines most commonly used as hosts in BICS are derived from the cabbage bollworm, Trichoplusia ni (Tn), or armyworm, Spodoptera frugiperda (Sf), and most of the biologics made with BICS are produced using the latter. The original Sf cell line, designated IPLB-SF-21, also known as Sf-21, was obtained from the ovaries of pupae in 1977. Other commonly used Sf cell lines include Sf9 (a subclone of IPLB-SF-21) and its subclones. derivatives, including Super 9 and Sf900+, also known as EXPRESSF+®. The original Tn cell line, designated TN-368, was obtained from ovarian tissue isolated from newly emerged virgin female moths, as reported by Hink in 1970. Other commonly used Tn cell lines include BTI-Tn-5B1-4 (marketed as HIGH FIVE ™) and Tni PRO cells.

In 2007, a group of scientists from Japan and New Zealand discovered that BTI-Tn-5B1-4 cells were contaminated with a new nodavirus (Li et al., J. Virol. 81:10890-96), referred to herein as memory "Tnnodavirus". This finding was confirmed and extended when all of our laboratory Tn cell lines, including TN-368, BTI-Tn-5B1-4, and Tni PRO, were found to be contaminated with this virus. Subsequently, in 2014, scientists at the US FDA's Center for Biologics Evaluation and Research (CBER) found that all Sf cell lines tested, including Sf-21 and Sf9 cells obtained from two reputable commercial sources , were contaminated with a rhabdovirus, now known as Sf-rhabdovirus (Ma et al., J. Virol. 88: 6576-85, 2014). A Takeda Vaccines, Inc. research group independently confirmed the presence of Sf-rhabdovirus in Sf9 cells used to produce their norovirus vaccine candidate (Takeda Vaccines, Inc., US Patent Application Publication No. US 2016/0244487; PCT/US14/59060 ). In addition, all of our laboratory Sf cell lines, including Sf-21, Sf9, and EXPRESSF+ ® , obtained from a variety of sources, were found to be contaminated with this virus.

There is a need for cell lines that are free of contaminating viruses and for methods of generating virus-free cell lines obtained from cell lines infected with viruses or organisms that are persistently infected or contain an endogenous virus.

JL Vaughn et al: "The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae)", In Vitro vol. 13, no. 4, April 1, 1977 describes a method for establishing Spodoptera frugiperda cell lines from virus-free cells. A. Saulnier et al: "Complete Cure of Persistent Virus Infections by Antiviral siRNAs", Molecular Therapy: The Journal Of The American Society of Gene Therapy, vol. 13, no. 1, January 1, 2006 describes a method of curing persistent poliovirus infection in HEp-2 cells using siRNA.

Summary

The present invention relates to a method for obtaining a Spodoptera frugiperda cell line free of Sfrhabdovirus as defined in independent claim 1.

Current teachings are directed to established cell lines derived from virus-contaminated cells or organisms, where the cell line is characterized by a lack of virus while retaining relevant cellular functions. Such established cell lines are particularly useful as components of biological platforms used for the production of vaccines, recombinant proteins, and biologicals for human and veterinary use, eg, BICS. Current teachings are also directed to methods of obtaining established virus-free cell lines from virus-contaminated cells or organisms that are virus-contaminated, eg, but not limited to, persistent infection or due to an endogenous virus.

According to an exemplary embodiment, an established insect cell line of the armyworm, Spodoptera frugiperda, is obtained directly or indirectly. This cell line is characterized by the lack of Sf-rhabdovirus, unlike Sf9 cells and the fall armyworm, from which the Sf9 cell line was derived. When compared to the Sf9 cell line, cells of this exemplary established cell line: grow to the same or very similar cell densities in culture, have the same or very similar cell diameters (size), have the same or very similar doubling times (rate growth), and produce similar patterns of W-glycosylation. Cells of this new established cell line are functionally different from Sf9 cells in that they produce more infectious recombinant baculovirus particles with AcP(-)p6.9hEPO or AcP(-)p6.9hSEAP and are not contaminated with Sfrhabdovirus. This new established cell line is further characterized as being structurally (genetically) different from Spodoptera frugiperda and from the Sf-21 cells, from which the Sf9 cells were derived.

In accordance with certain embodiments of cell lines, an exemplary established cell line characterized by lack of virus is obtained from a virus-contaminated organism or from virus-contaminated cells. In certain examples disclosed herein, the cell line is derived from a virus-contaminated Ni Trichoplusia cell. In certain examples described herein, the Trichoplusia ni cell line is the TN-368 cell line. In certain examples described herein, the virus is an alphanodavirus. As also described herein, the cell line is further characterized by a cell density, average cell diameter, morphology, and W-glycosylation pattern that is the same or substantially the same as TN-368 cells when cell line and TN-368 cells are propagated under the same conditions.

Also described herein is another exemplary established cell line, an insect cell line that is derived directly or indirectly from the cabbage looper, Trichoplusia ni. This cell line is characterized by the lack of nodavirus, unlike TN-368 cells, derived from Trichoplusia ni. When compared to the TN-368 cell line, cells of this established exemplary cell line: grow to the same or very similar cell densities in culture, have the same or very similar cell diameters (size), and produce n-glycosylation patterns.

According to an exemplary method for obtaining a virus-free cell line derived from a virus-contaminated organism or virus-contaminated cells, comprises: isolating a cell from a virus-contaminated organism or from virus-contaminated cells; combining the isolated cell with a cell culture medium comprising an antiviral compound to form a first culture composition; incubating the first culture composition under conditions suitable for the cell to grow and divide, thereby generating a multiplicity of cells; removing a portion of the multiplicity of cells or cell culture medium and testing for the presence or absence of a virus; combining at least part of the multiplicity of cells with cell culture media without an antiviral compound to form a second culture composition; and incubating the second culture composition under suitable conditions for the cells to grow and divide, thus obtaining a virus-free cell line.

According to certain exemplary methods, an established cell line is obtained by isolating a single cell or a small number of cells from virus-infected primary cells, a virus-contaminated cell line, or a contaminated organism. The isolated cell(s) are combined with cell culture media containing an antiviral compound, forming a first culture composition. The first culture composition is incubated in conditions that allow cells to grow and divide, thus generating a multiplicity of cells. Culture media are periodically replaced with fresh culture media containing the antiviral compound. A small number of cells obtained from the first culture composition or a volume of culture medium obtained from the first culture composition is tested for the presence or absence of virus. When no viral nucleic acid is detected, at least some of the cells in the first culture composition are combined with culture media without the antiviral compound to form a second culture composition. The second culture composition is incubated under conditions that allow cells to grow and divide, and the culture medium is periodically replaced with fresh culture medium. In certain embodiments, as the cells continue to grow, the number of cells increases and the cells expand from one culture vessel to multiple vessels, even as the cells divide periodically (also known as passage). In certain embodiments, a sample of cells or culture media from at least one culture vessel is analyzed for the presence or absence of viral nucleic acid, an indicator of the presence of viruses. Once the number of cells has reached a sufficient amount, aliquots of the cells can be frozen or stored using known methods.

Brief description of the figures

These and other features and advantages of the current teachings will become better understood with reference to the following description, the appended claims, and the accompanying figures. One skilled in the art will understand that the figures, described below, are for illustrative purposes only and are not intended to limit the scope of the teachings described in any way.

FIGS. 1A-1C: Sf-rhabdovirus in polyclonal Sf9 cells treated with antiviral drug. Polyclonal Sf9 cell populations were treated for approximately one month with various concentrations of ribavirin (FIGS. 1A and 1B) or ribavirin, 6-azauridine, and vidarabine (FIG. 1C), and then total RNA was extracted and analyzed for Sf-rhabdovirus RNA by RT-PCR, as described in Example 4. The results shown in FIGS. 1A and 1C were obtained using RNA from cells that were still being cultured in the presence of antiviral drugs, while those shown in FIG. 1B were obtained using RNAs from cells that had been treated with ribavirin, but then passaged 12 times in the absence of antiviral drugs, as described in Example 2. Total RNAs extracted from Drosophila melanogaster Sf9 cells or S2R+ cells ( "S2" in Figures 1A-1C, 2A-2B, 3A-3C) as positive and negative controls, respectively. An additional negative control reaction without template (H2O) was performed. Lanes marked "M" show the 100 bp markers, with selected sizes indicated on the left.

FIGS. 2A-2B: Sf-rhabdovirus in Sf9 cell subclones treated with isolated antiviral drugs. Individual Sf9 cell subclones were isolated by limiting dilution and treated for approximately one month with various antiviral drugs, and then total RNA was extracted from individual clones and Sfrhabdovirus RNA was analyzed by RT-PCr (FIG. 2A) or Rt-PCR, followed by nested PCR (FIG. 2B), as described in Example 4. All results shown in FIGS. 2A-2B were obtained using RNA from cells that were still being cultured in the presence of antiviral drugs. Positive and negative controls and 100 bp markers were as described in the brief description of FIGS. 1A-1C.

FIGS. 3A-3C: Absence of Sf-rhabdovirus in Sf-RVN cells. Total RNA was isolated from an example cell line, designated "Sf-RVN", at various passage levels and analyzed for the presence of Sf-rhabdovirus using Sf-rhabdovirus-specific RT-PCR, followed by nested PCR, as is described in Example 4. This exemplary cell line was generated by expanding a 6-azauridine-treated Sf9 subclone that was found to be negative for Sf-rhabdovirus contamination in FIG. 2B. The results of Sfrhabdovirus-specific nested RT-PCR/PCR demonstrated that there was no presence of Sf-rhabdovirus within 60 passages and 120 passages of Sf-RVN cells (Figures 3A and 3B, respectively). Total RNA was also isolated from the pellet fraction obtained by ultracentrifugation of cell-free media (CFM) from Sf-RVN cells at passage 60. Total RNA from this CFM pellet was analyzed for Sf-rhabdovirus using Sf-rhabdovirus-specific nested RT-PCR/PCR. As shown in FIG. 3C, an Sf-rhabdovirus amplicon was observed in lanes corresponding to RNA isolated from Sf9 cells and RNA isolated from Sf9 cell-free medium pellet (FIG.

3C, Sf9 and Sf9 CFM lanes, respectively). In contrast, the Sf-rhabdovirus amplicon was not detected in the RNA isolated from the Sf-RVN cell-free medium pellet (FIG. 3C, Sf-RVN CFM lane). All results shown in FIGS. 3A-3C were obtained using RNA from cells that were cultured in the absence of antiviral drugs. RNAs extracted from Sf9 cells and from the pellet obtained by Sf9 CFM ultracentrifugation were used as positive controls; and RNAs extracted from S2R+ (S2) cells were used as negative controls. An additional negative control reaction without template (H2O) was performed, and the lanes marked M show the 100 bp markers, with the selected sizes indicated on the left.

FIG. 4: Mycoplasma assays. Sf-RVN and Sf9(-) cell extracts were analyzed for mycoplasma contamination using the PCR-based universal mycoplasma detection kit (American Type Culture Collection), as described in Example 6. A plasmid was used that encodes an M. arginini rRNA target sequence as a positive control (FIG. 4, lane "M. arginini"). Additional controls were performed using Sf-RVN and Sf9 cell lysates spiked with this plasmid (FIG. 4, lanes labeled as Sf-RVN (+) and Sf9 (+), respectively) to determine if the lysate interfered with the assay.A negative control reaction was performed without mold (FIG. 4, lane H2O). The lane marked M shows the 100 bp markers, with the selected sizes indicated on the left.

FIGS. 5A-5D: Spodoptera cell culture and morphology. Sf-RVN and Sf9 cells were seeded in shake flasks at a density of 1.0 x 106 cells/mL in ESF 921 medium. Triplicate samples were collected at various time points after seeding and viable cell counts were measured. and diameters with an automated cell counter, as described in Example 7. The figure shows the average viable cell densities (FIG. 5A), diameters (FIG. 5B), and doubling times (FIG. 5C) measured. in three independent experiments, as well as phase contrast micrographs of Sf-RVN and Sf9 cells at 10X magnification, FIG. 5 D). Error bars represent confidence intervals (P<0.05).

FIGS. 6A-6B: Cell viability after baculovirus infection. Sf-RVN and Sf9 cells were infected with a free Sf-rhabdovirus stock of AcP(-)p6.9hSEAp at an MOI of 0.1 cfu/cell (FIG. 6a) or 5 cfu/cell (FIG.

6B). Triplicate samples were collected at various time points after infection and viability was measured using an automated cell counter, as described in Example 7. The graphs show the mean percent viability measured in two independent experiments. Error bars represent confidence intervals (P<0.05).

FIGS. 7A-7C: Production of recombinant p-gal. Sf-RVN and Sf9 cells were infected with a BacPAK6-AChi/Cath Sfrhabdovirus-free stock at an MOI of 5 cfu/cell. Triplicate samples were collected at various time points post-infection and clarified intracellular extracts were analyzed for p-gal activity (FIG. 7A), as described in Example 9. This graph shows the average results with bars of error represented by the confidence intervals (P<0.05). A pool of extracts was also used to measure total intracellular p-gal production levels by immunoblot analysis (FIG. 7B) with scanning laser densitometry (FIG. 7C) to estimate the relative densities of immunoreactive bands. The same general trends were observed in two independent biological replicates of this experiment.

FIGS. 8A-8F: Production of recombinant hSEAP. Sf-RVN and Sf9 cells were infected with an Sf-rhabdovirus-free stock of AcP(-)p6.9hSEAP at an MOI of 0.1 pfu/cell (FIGS. 8A, 8B and 8C) or 5 pfu/cell ( FIGS 8D, 8E and 8F). Triplicate samples were collected at various time points post-infection, cell-free media were prepared and assayed for hSEAP activity, as described in Example 9, and average results were plotted with error bars representing confidence intervals (P<0.05; FIGS. 8A and 8D). A cell-free media pool was also used to measure total extracellular hSEAP production levels by immunoblot analysis (FIGS. 8B and 8E) with scanning laser densitometry (FIGS. 8C and 8F) to estimate the relative densities of bands. immunoreactive.

FIGS. 9A-9B: Production of recombinant hEPO. Sf-RVN and Sf9 cells were infected with a Sfrhabdovirus-free stock of AcP(-)p6.9hEPO at an MOI of 5 cfu/cell. Samples were collected at various time points after infection and cell-free media were prepared and analyzed for levels of total extracellular hEPO production by immunoblot analysis (FIG. 9A), with scanning laser densitometry (FIG.

9B) to estimate the relative densities of the immunoreactive bands, as described in Example 9.

FIGS. 10A-10B: N-glycosylation profiles. Sf-RVN and Sf9 cells were infected with a Sfrhabdovirus-free stock of AcP(-)p6.9hEPO at an MOI of 3 pfu/cell, and hEPO-His was affinity purified from cell-free medium, as described in Example 10. W-glycans were enzymatically released, recovered, permethylated and analyzed by MALDI-TOF MS (FIG. 10A), according to known methods, with molecular ions detected as assigned structures [M Na] +, annotated using the symbolic representations in standard bullets, numbered for simplicity and presented as percentages of the total (FIG. 10B).

FIG. 11: Production of recombinant baculoviruses. Sf-RVN and Sf9 cells were infected with Sfrhabdovirus-free stocks of AcP(-)p6.9hSEAP or AcP(-)p6.9hEPO. The resulting progeny were collected and titered by plaque assays, as described in Example 11. The resulting titers were plotted as the mean viral titers obtained in three independent experiments, with error bars representing confidence intervals plotted as ( P<0.05) or '**' (P<0.001).

FIG. 12: Sf-rhabdovirus infection of Sf-RVN cells. Sf-RVN cells were mock-infected or infected with cell-free medium derived from Sf9 cells, as described in Example 13, and then total RNA was extracted and analyzed for Sf-rhabdovirus by RT- PCR, as described in Example 4. Lane marked: M contains base pair markers; Sf9 contains material amplified from RNA from Sf9 cells; mock contains material amplified from RNA of Sf-RVN cells obtained after cells were mock infected; P0 (24) and (72) contain material amplified from RNA of Sf-RVN cells obtained after cells were infected with Sf-rhabdovirus for 24 and 72 h, respectively; P1 (72), P2 (72), and P3 (72) contain material amplified from RNA of Sf-RVN cells obtained 72 h after the first, second, or third passage of cells after infection with Sf-rhabdovirus , respectively; S2 contains material amplified from RNA from S2R+ cells; and H2O contains distilled water.

FIGS. 13A-13B: Tn-nodavirus in polyclonal TN-368 cells treated with antiviral drugs. TN-368 polyclonal cell populations were treated for 15 days with various concentrations of a cocktail of three antiviral drugs, ribavirin, 6-azauridine, and vidarabine. Total RNA was extracted from cells still growing in the presence of these drugs and analyzed for segment 1 (FIG. 13A) or 2 (FIG. 13B) of Tn nodavirus RNA by RT-PCR, as shown. described in Example 16. Total RNAs extracted from TN-368 or Sf9 cells were used as positive or negative controls, respectively. An additional negative control reaction without template (H2O) was performed. Lanes marked "M" show the 100 bp markers, with selected sizes indicated on the left.

FIGS. 14A-14B: Absence of Tn-nodavirus in subclones of TN-368 cells treated with ribavirin. Total RNA was isolated from six single cell TN-368 subclones treated for one month with 200 pg/ml ribavirin. Samples were then analyzed for Tn nodavirus RNA segment 1 by RT-PCR (FIG. 14A) or RT-PCR, followed by nested PCR (FIG. 14B), as described in Example 16. Total RNAs extracted from TN-368 or Sf9 cells were used as positive or negative controls, respectively. Lanes marked "M" show the 100 bp markers, with selected sizes indicated on the left.

FIGS. 15A-15C: Absence of Tn-nodavirus in Tn-NVN cells. Total RNA was isolated from an exemplary cell line, designated "Tn-NVN", cultured at various passages in the absence of antiviral drugs and analyzed for the presence of Tnnodavirus by RT-PCR, followed by nested PCR with segment 1-specific primers ( FIG. 15A) or segment 2 (FIG. 15B) of Tn-nodavirus, as described in Example 16. This exemplary cell line, designated Tn-NVN, was generated by expanding a clone of TN-368 treated with ribavirin (Cl #3), which was found to be negative for Tn-nodavirus contamination (Figures 15A-15C). The results of Tnnodavirus-specific RT-PCR, followed by nested PCR, showed that no Tn-nodavirus was present after 55 passages of Tn-NVN cells (Figures 15A and 15B). Total RNA was also isolated from the pellet fraction obtained by ultracentrifuging the CFM of Tn-NVN cells at passage 55 and used to analyze Tn-nodavirus using Tn-nodavirus-specific RT-PCR, followed by nested PCR. , as described in Example 16. As shown in FIG.

15C, a Tn-nodavirus amplicon was observed in lanes corresponding to RNA isolated from TN-368 cells and RNA isolated from the TN-368 cell-free media pellet (FIG. 15C, lanes TN-368 and TN-368 ^c F ^m , respectively). In contrast, the Tn-nodavirus amplicon was not detected in the RNA isolated from the Tn-NVN cell-free media pellet. Again, all of the results shown in FIG. 15A-15C were obtained using RNA from cells cultured in the absence of antiviral drugs. RNAs extracted from TN-368 cells and from the pellet obtained by ultracentrifugation of TN-368 cell-free media (CFM) were used as positive controls, and RNA extracted from Sf9 cells was used as negative control. An additional negative control reaction without template (H2O) was performed, and the lanes marked M show the 100 bp markers, with the selected sizes indicated on the left.

FIG. 16: Mycoplasma assays. Tn-NVN and TN-368 (-) cell extracts were analyzed for mycoplasma contamination using the PCR-based universal mycoplasma detection kit (American Type Culture Collection), as described in Examples 6 and 18. A plasmid encoding an M. arginini rRNA target sequence was used as a positive control (FIG. 16, lane 'M. arginini'). Additional controls were performed using Tn-NVN and TN-368 cell lysates spiked with this plasmid. (FIG. 16, lanes Tn-NVN (+) and TN-368 (+), respectively) to determine if the lysate interfered with the assay. A negative control reaction without template (H2O) was performed. The lane marked with M shows the 100 bp markers, with the selected sizes indicated on the left.

FIGS. 17A-17C: Culture and cell morphology. Tn-NVN and TN-368 cells were seeded in shake flasks at a density of 1.0 x 106 cells/mL in ESF 921 medium. Triplicate samples were collected at various time points after seeding and cell counts were measured. viable cells and diameters with an automated cell counter, as described in Examples 7 and 19. The figure represents the average viable cell densities (FIG. 17A) and diameters (FIG. 17B) measured in three independent experiments, as well as phase contrast micrographs of Tn-NVN and TN-368 cells at 10X magnification (FIG. 17C). Error bars represent confidence intervals (P<0.05).

FIGS. 18A-18C: Production of recombinant p-gal. Tn-NVN and TN-368 cells were infected with a free Tn-nodavirus stock of BacPAK6-AChi/Cath at an MOI of 5 cfu/cell. Triplicate samples were collected at various time points after infection and clarified intracellular extracts were analyzed for p-gal activity (FIG.

18A), as described in Examples 9 and 20. This graph shows the average results with error bars representing confidence intervals (P<0.05). A pool of extracts was also used to measure total intracellular p-gal production levels by immunoblot analysis (FIG. 18B), with scanning laser densitometry (FIG. 18C) to estimate relative densities of immunoreactive bands. The same general trends were observed in two independent biological replicates of this experiment.

FIGS. 19A-19C: Production of recombinant hSEAP. Tn-NVN and TN-368 cells were infected with a Tn-nodavirus-free stock of AcP(-)p6.9hSEAP at an MOI of 5 cfu/cell. Triplicate samples were collected at various time points post-infection, cell-free media were prepared and assayed for hSEAP activity, as described in Examples 9 and 20, and average results were plotted with error bars. represent confidence intervals (P<0.05; FIG. 19A). A set of free media was also used of cells to measure levels of total extracellular hSEAP production by immunoblot analysis (FIG. 19B), with scanning laser densitometry (FIG. 19C) to estimate relative immunoreactive band densities.

FIGS. 20A-20B: Production of recombinant hEPO. Tn-NVN and TN-368 cells were infected with a Tn-nodavirus-free stock of AcP(-)p6.9hEPO at an MOI of 5 cfu/cell. Samples were collected at various time points after infection and cell-free media were prepared and analyzed for levels of total extracellular hEPO production by immunoblot analysis (FIG. 20A), with scanning laser densitometry (FIG. 20B). ) used to estimate the relative densities of immunoreactive bands, as described in Examples 9 and 20.

FIGS. 21A-21B: W-glycosylation profiles. Tn-NVN and TN-368 cells were infected with a Tnnodavirus-free stock of AcP(-)p6.9hEPO at an MOI of 3 pfu/cell and hEPO-His was affinity purified from cell-free medium, as is described in Examples 10 and 21. The W-glycans were enzymatically released, recovered, permethylated and analyzed by MALDI-TOF MS (FIG. 21A), according to known methods, and the molecular ions detected as [M Na] + Structures were assigned, annotated using standard vignette symbolic representations, numbered for simplicity, and presented as percentages of the total (FIG. 21B).

FIG. 22: Sf-rhabdovirus in BmN cells. Total RNA was extracted from the BmN cell line, derived from the lepidopteran insect, Bombyx mori, as described in Example 4. Samples were then analyzed for various Sf-rhabdovirus RNAs (N, P, M, G , X and L) by RT-PCR, as described in Example 22.

Detailed Description of Certain Exemplary Embodiments

It is to be understood that both the foregoing general description and the following detailed descriptions are illustrative and exemplary only, and are not intended to limit the scope of the disclosed teachings. The section headings used herein are for organizational purposes only, and should not be construed as limiting the subject matter of the teachings described.

In the foregoing Summary, Detailed Description, accompanying Figures, and claims below, reference is made to particular features (including method steps) of the current teachings. The description in this specification is to be understood to include the possible combinations of such particular features. For example, but without limitation, when a particular feature is described in the context of a particular embodiment of the current teachings, or a particular claim, that feature may also be used, to the extent possible, in combination with and/or in the context of other particular embodiments, and in current teachings in general.

Where reference is made to a method comprising two or more steps in combination, the defined steps may be performed in any order or simultaneously (except where the context precludes that possibility), and the method may include one or more additional steps that are performed before any of the defined stages, between two of the defined stages, or after all of the defined stages (except where the context excludes that possibility).

Definitions

The term "cell line", used in reference to current teachings, means a population of cells that expanded from one or a few common predecessor cells, for example, but not limited to, a clonal population of cells that expanded to from a single isolated cell. An "established cell line" is a cell line that has the potential to proliferate indefinitely when provided with fresh culture medium, room to grow, and when incubated under suitable conditions. These cell lines have undergone changes in vitro (eg, but not limited to, transformation, chromosomal changes, or both) compared to the natural cell counterpart found in the body. A cell line that is obtained by isolating a single cell from a first cell line and then expanding the isolated cell to a multiplicity of cells to obtain a second cell line is sometimes called a "subclone" of the first cell line from which it was derived. .

As used herein, the term "comprising", which is synonymous with "including" or "characterized by", and cognates of each (such as comprises and includes), is inclusive or open-ended, and does not exclude components. , elements, or method steps, ie, other components, steps, etc., are optionally present. For example, but not limited to, an article that "comprises" components A, B, and C may consist of (ie, contain only) components A, B, and C; or the article may contain not only components A, B and C, but also one or more additional components.

As used herein, the term "derived" means obtained from a source, directly or indirectly. For example, cells can be derived directly from an organism by obtaining a tissue or organ from the organism and then disaggregating the tissue or organ to obtain primary cells. Cells can be obtained indirectly from an organism, for example, but not limited to, by obtaining an isolate, typically a single cell isolate, from a cell line that was obtained from the organism, then expanding the isolate to obtain a cell line comprising a multiplicity of cells, sometimes called subclones.

The term "lepidoptera insect" refers to any member of a large order (Lepidoptera) of insects comprising butterflies, moths and grasshoppers which, as adults, have four broad or lanceolate wings, usually covered with minute overlapping scales and often , brightly colored and as larvae are caterpillars. Exemplary lepidopteran insects include, but are not limited to, Spodoptera frugiperda, Bombyx mori, Heliothis subflexa, and Trichoplusia ni.

As used herein, the term "substantially" refers to a variation of no more or less than ten percent relative to the item(s) mentioned. For example, but not limited to, a cell line that has an average cell diameter that is between 90% and 110% of the average diameter of Sf9 cells, over a statistically significant sample size, when the cell line and cells Sf9 are propagated under the same conditions, and the average cell diameter is determined as described herein; or a cell line that has a cell density that is between 90% and 110% of the cell density of Sf9 cells, over a statistically significant sample size, when the cell line and Sf9 cells are propagated in the same conditions, and cell density is determined as described herein.

The expressions "test for the presence of viruses", "test for the presence of Sf-rhabdovirus", "test for the presence of Tn-nodavirus", "detection of the presence or absence of viruses" and related terminology are used in a broad sense herein. Those skilled in the art understand that there are numerous testing techniques known in the art that can be employed in the context of the current teachings. Examples of techniques suitable for testing for the presence of viruses include reverse transcription (RT), RT-polymerase chain reaction (RT-PCR), RT-PCR in conjunction with nested PCR (for example, but not limited to the techniques examples described in Examples 4, 16, and 22), quantitative PCR (sometimes referred to as real-time PCR), various probe hybridization techniques, electron microscopy, and various antibody-based detection techniques known in the art, for example, but without limitation, an ELISA assay comprising at least one anti-virus antibody. In the case of a virus that is lytic or causes an observable cytopathic effect (CPE) in the cell, exemplary testing techniques include, but are not limited to, plaque assay and CPE observation, which may comprise the use of microscopy. Bioinformatic techniques, eg, but not limited to, BLAST searchable electronic databases of RNA or DNA sequences contained in cell lines or organisms of interest, are also within the scope of the current teachings.

According to certain embodiments, an established cell line characterized by a lack of virus may be derived from an organism that is infected with a virus, for example, but not limited to, an established virus-free cell line derived from an organism contaminated with viruses. In the present invention, the cell line is derived from Spodoptera frugiperda (Sf). Also described herein are cell lines derived from an insect contaminated with a virus. Also described herein are insect-derived cell lines in which the insect comprises a lepidopteran insect, for example, but not limited to, Bombyx mori, Heliothis subfloxa, or Trichoplusia ni. In certain embodiments, the cell line is derived from an Sf cell line, for example, but not limited to, the Sf9 or Sf-21 cell lines. Also described herein are cell lines derived from Trichoplusia not contaminated with a virus or a Trichoplusia cell line not contaminated with a virus, for example, but not limited to, the TN-368 cell line contaminated with an alphanodavirus.

As described herein, an established cell line is characterized as having the same or substantially the same cell density, doubling time, average cell diameter, morphology, and W-glycosylation pattern as the virus-contaminated cells from which it was derived. cell line, when: (1) virus-infected and virus-free cell lines are propagated under the same conditions, (2) comparison is made as described herein, and (3) comparisons are based on a statistically significant sample size. As described herein, the cell lines are characterized by the production of more infectious recombinant baculoviruses than the virus-infected cells from which the cell line was derived, when each is infected with AcP(-)p6.9hEPO or AcP (-)p6.9hSEAP under the same conditions, and the comparison is made according to Example 11. As described herein, the cell lines of the current teachings are susceptible to infection by Sf-rhabdovirus.

In accordance with certain exemplary methods for obtaining cell lines lacking viruses, one or a few cells are isolated from a population of cells that are infected with viruses, such as a cell line that is contaminated with a virus or cells from a tissue or organ. disaggregated or from an organism that is infected with a virus. The isolated cell or cells are combined with an appropriate cell culture medium containing one or more antiviral compounds to form a first culture composition. This first culture composition is incubated under conditions suitable for cells to grow and divide; and for a period of time sufficient to allow one or more antiviral compounds to affect viral replication. In certain embodiments of the method, an aliquot of the cells or culture medium is removed from the culture and tested for the presence of virus. Cells lacking virus are combined with culture media that do not contain an antiviral compound to form a second culture composition. This second culture composition is incubated under conditions that allow the cells to grow and divide. The cells are expanded to obtain a cell line that lacks the virus that contaminated the organism or cells from which the cell line was derived.

In certain embodiments, methods of obtaining a virus-free cell line comprise: isolating a single cell from a population of Spodoptera frugiperda insect cells; combine the isolated cell with culture media cells comprising at least one antiviral compound to form a first culture composition; incubating the first culture composition under conditions suitable for the cell to grow and divide, thereby generating a multiplicity of cells; optionally removing a portion of the cells or cell culture medium and testing for the presence of Sf-rhabdovirus or a nodavirus; combining at least part of the multiplicity of cells in the first culture composition with cell culture media without an antiviral compound to form a second culture composition; and incubating the second culture composition under suitable conditions for the cells to grow and divide, thus obtaining a cell line characterized by the absence of viruses.

According to certain embodiments of the method, established cell lines lacking Sfrhabdovirus are obtained. As described herein, an individual cell or small groups of cells, for example, but not limited to groups of 2 cells, 3 cells, 4 cells, 5 cells, 10 cells or less, or 20 cells or less (including each integer between 1 and 20) are isolated from a population of cells that is infected with a virus. Non-limiting examples of techniques to isolate a single cell or small numbers of cells include cloning by limiting dilution (sometimes referred to as serial dilution cloning), cloning of cells in soft agar, and subsequent cell colony selection. , cell sorting to isolate single or small numbers of cells, laser capture microdissection (LCM), use of micropipettes (eg, but not limited to, ultra-thin capillaries) to manually capture single or small numbers of cells, microfluidic techniques, or use of micromanipulators to microscopically assist in the selection of individual or small numbers of cells. In certain embodiments, single cell isolation comprises cloning by limited dilution.

In certain embodiments of the method, isolated single cells or small groups of cells are combined with cell culture media comprising at least one antiviral compound to form a first culture composition. Examples of antiviral compounds include, but are not limited to, drugs such as nucleoside analogs, interferon, and viral-specific antibodies, for example, but not limited to, neutralizing monoclonal or polyclonal antibodies. Non-limiting examples of nucleoside analogs include ribavirin, 6-azauridine, vidarabine, acyclovir, 9-/3-D-arabinofuranosyladenine (Ara-A), cytosine arabinose, adenine arabinoside, and Guanine 7-N-oxide (G-7- ox). In certain embodiments of the method, the at least one antiviral compound comprises 6-azauridine. In certain embodiments, the antiviral compound is selected from ribavirin, 6-azauridine, vidarabine, acyclovir, 9-/3-D-arabinofuranosyladenine (Ara-A), cytosine arabinose, adenine arabinoside, and 7-N-guanine oxide (G -7-Ox). In certain embodiments, the antiviral compound is 6-azauridine. In certain embodiments, the antiviral compound comprises ribavirin.

In accordance with certain embodiments of the method, the first culture composition is incubated under conditions suitable for cell growth. In accordance with certain disclosed methods, cells or cell culture supernatant obtained from the first culture composition are analyzed for the presence or absence of viruses, for example, but not limited to, RT-PCR, nested PCR, or RT-PCR. and nested PCR, followed by analysis of the resulting amplicons for the presence or absence of virus-specific amplification products. In certain embodiments of the method, the presence or absence of infectious virus is determined by: (1) combining (a) potentially infected cells or cell culture supernatant in which potentially infected cells were incubated with (b) cells that are susceptible to infection by the potential virus (c) in a suitable cell culture medium; (2) incubate this culture under conditions suitable for the virus to infect the cells; and (3) monitoring the cultured cells or the media in which they have been cultured for the presence of viral nucleic acid. In certain embodiments, the cells or culture media are periodically tested for the presence of viruses, for example, but not limited to, by use of virus-specific RT-PCR followed by nested PCR, and then the presence is determined. or absence of specific amplicons.

According to certain embodiments, after the isolated cells have been incubated in the first culture composition for a period adequate to inhibit viral replication and samples of the corresponding cells or culture media have been analyzed and determined not to contain virus, the cells are combined with cell culture medium that does not contain an antiviral compound to form a second culture composition. The second culture composition is incubated under conditions suitable for cell growth. In certain embodiments, the cells or culture media of the second culture composition are analyzed for the presence or absence of virus.

Those skilled in the art will appreciate that suitable conditions for culturing a particular cell type can be readily determined from a variety of sources, for example, but not limited to, cell culture manuals, commercial cell banks, or cell media suppliers. crop and/or plastic items. Appropriate cell culture conditions can also be readily determined using methods known in the art.

In certain embodiments, the cell lines are derived from primary cells that are contaminated with Sf-rhabdovirus. In certain embodiments, the cell lines are derived from a cell line that is infected with Sf-rhabdovirus. In certain embodiments, the infected cell population is part of an infected cell line. In certain embodiments, the infected cell line was obtained from an infected organism, eg, but not limited to moths, caterpillars, or other insects that are persistently infected with a virus. In certain embodiments, the cell line is derived from a contaminated cell line that is derived from a virus-infected insect, for example, but not limited to, Sf-rhabdovirus-infected Sf cell lines. Also described herein are cell lines which are a Trichoplusia cell line neither contaminated with Tn-nodavirus, for example, but not limited to, TN-368, BTI-Tn-5B1-4 (also known as HIGH FIVE™), or Tni PRO cells.

In accordance with certain disclosed methods, the cells or cell culture media in which the cells were grown are analyzed for the presence or absence of viruses. In certain methods, the test comprises RT-PCR and nested PCR; quantitative PCR; probe hybridization techniques; bioinformatic methods including, but not limited to, BLAST search; plate assay, CPE observation, or antibody-based detection methods.

Certain Exemplary Embodiments

Example 1. Insect cell culture. Sf9 cells, known to be contaminated with Sf-rhabdovirus, were routinely maintained as shake flask cultures at 28°C in ESF 921 medium (Expression Systems, Woodland, CA). TN-368 cells, known to be contaminated with Tn-nodaviruses, were routinely maintained as adherent cultures at 28°C in TN-MFH medium supplemented with 10% fetal bovine serum (Atlanta Biologicals, Inc., Flowery Branch, GA) and 1% Pluronic F-68 (Invitrogen, Carlsbad, CA).

Example 2. Conventional methodology is not capable of producing an established S. frugiperda cell line lacking virus. Initial efforts to isolate a free derivative of Sf-rhabdovirus involved culturing polyclonal Sf9 cell populations in TNM-FH medium supplemented with 10% (v/v) fetal bovine serum (Atlanta Biologicals, Inc., Flowery Branch, GA ) plus various concentrations of ribavirin (Oxchem Corporation, Irwindale, CA). Subsequently, we treated populations of polyclonal Sf9 cells with various concentrations of three antiviral drugs, ribavirin, 6-azauridine (Alfa Aesar, Ward Hill, MA), and vidarabine (TCI America, Portland, OR). Sf9 cells were cultured with these three drugs for approximately one month with custom serial passages and samples were routinely analyzed for Sf-rhabdovirus contamination by RT-PCR, as described in Example 4. After After approximately one month of treatment with 100 pg/mL ribavirin, an Sf9 subclone was obtained that did not contain Sf-rhabdovirus detectable by RT-PCR (Figure 1A). This Sfrhabdovirus-free subclone was transferred to TNM-FH medium supplemented with 10% (v/v) fetal bovine serum, but without antiviral drugs, and re-analyzed by RT-PCR/nested PCR, as described in Example 4. Surprisingly, when these cells were transferred to culture medium lacking antiviral drugs, they reverted to the Sf-rhabdovirus-positive phenotype (Figure 1B). Subsequently, populations of polyclonal Sf9 cells were treated with various concentrations of a combination of three antiviral drugs, ribavirin, 6-azauridine, and vidarabine. Again, it was surprising to find that cells treated with these three drugs for approximately one month remained positive for Sf-rhabdovirus (FIG. 1C). Thus, in stark contrast to previous work, in which vertebrate cells were cured of rhabdoviral contamination by treatment with these same antiviral drugs, this approach failed to eliminate Sf-rhabdovirus from Sf9 cells.

Example 3. Exemplary method for obtaining an established Sf cell line characterized by the lack of virus. After discovering that polyclonal Sf9 cell cultures treated with antiviral drugs reverted to the positive Sf-rhabdovirus phenotype when grown in drug-free media, novel methods were developed to obtain established virus-free cell lines derived from virus-contaminated starting material. This exemplary embodiment comprised isolating individual Sf9 cells by limiting dilution and then treating subclones of isolated cells with antiviral drugs. Cells were seeded in 96-well plates in TNM-FH medium supplemented with 10% (v/v) fetal bovine serum (Atlanta Biologicals, Inc., Flowery Branch, GA) plus 10 pg/mL ribavirin (Oxchem Corporation, Irwindale, CA), 6-azauridine (Alfa Aesar, Ward Hill, MA), or vidarabine (TCI America, Portland, OR). Cells were cultured for approximately one month with custom amplification to produce progressively larger cultures, and after reaching the 25 cm2 flask level, samples were tested for Sf-rhabdovirus contamination by PCR, as described in Example 4. A clone lacking Sf-rhabdovirus contamination (FIG. 2B) was transferred to media lacking antiviral drugs, designated Sf-RVN zero passage (P0) and, at P2, transferred to culture in shaken flask in ESF 921 medium without serum and subsequently maintained in this medium and culture format.

Example 4. Sf-rhabdovirus-specific reverse transcription PCR (RT-PCR)/Nested PCR. Samples of Sf9 and Sf-RVN cultures containing 1 x 10 6 cells were collected, and the cells pelleted by low speed centrifugation. Cell-free supernatants were filtered through a 0.22 pm filter (CELLTREAT Scientific, Shirley, MA) and then ultracentrifuged at 131,000 xg for 22 h at 4°C. Total RNA was extracted from both low-speed cell-bearing pellets and high-speed cell-free pellets using RNASo/y reagent (Omega Bio-Tek, Inc., Norcross, GA), according to the manufacturer's protocol. RNAs were then quantified and used as templates for cDNA synthesis with the ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA) and an Sf-rhabdovirus-specific primer designated 320-SP1 (SEQ ID NO: 9), according to the manufacturer's protocol. Equivalent amounts of each cDNA preparation were used for PCRs with Taq DNA polymerase, ThermoPol reaction buffer (New England Biolabs), and Sfrhabdovirus Mono-1 (SEQ ID NO: 1) and Mono-2 (SEQ ID NO: 1) specific primers. two). The reaction mixtures were incubated at 94 °C for 3 min, cycled 35 times at 94 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min, and finally incubated at 72 °C for 10 s. min. One pL of each primary PCR (RT-PCR) was then used as a template for the secondary PCRs (RT-PCR/nested PCR) under the same conditions, except that the primers were primers specific nests of Sf-rhabdovirus Mono-1i (SEQ ID NO: 7) and Mono-2i (SEQ ID NO: 8). RT-PCR and RT-PCR followed by nested PCR products were analyzed by ethidium bromide-stained agarose gel electrophoresis according to standard methodology. The sequence of each primer used for these assays is shown in Table 1.

Example 5. The exemplary Sf subclone "Sf-RVN" lacks Sf-rhabdovirus. Total RNA was isolated from Sf-RVN cell extracts at various passage levels and analyzed for the presence of Sf-rhabdovirus by RT-PCR/nested PCR, as described in Example 4. A strong amplification product of the expected size when total RNA from Sf9 cells was used as a positive control for this assay, as expected (Figures 3A, 3B and 3C). In contrast, no products were observed when total RNAs isolated from Sf-RVN cells were used every five passages over the course of 60 (FIG. 3A) or 120 (FIG. 3B) sequential passages in the absence of any antiviral drugs in the laboratory. . Neither were amplification products observed in negative controls with total RNA isolated from D. melanogaster S2R+ cells, which do not support Sf-rhabdovirus replication. Strong amplification products of the expected sizes were observed when using two other Sf-rhabdovirus-specific primer pairs (Mono-3 (SEQ ID N2: 3)/Mono-4 (SEQ ID N2: 4) and Mono-5 ( SEQ ID N2: 4) N.2: 5)/Mono-6 (SEQ ID N2: 6); see Table 1), which were derived from other regions of the Sf-rhabdovirus L protein coding sequence, for RT-PCR with total RNA isolated from Sf9 cells, but not from Sf-RVN cells (data not shown). Finally, we observed a strong amplification product of the expected size in RT-PCR/Nested PCR assays with total RNA isolated from pellets obtained by ultracentrifugation of Sf9 cell-free media, but not Sf-RVN cell-free media, when analyzed after 60 passes (FIG. 3C). Together, these results demonstrated that there was no detectable Sf-rhabdovirus RNA in Sf-RVN cells or cell-free media over the course of 120 passages in the absence of antiviral drugs, indicating that these cells are free of Sf-rhabdovirus. .

Example 6. Detection of mycoplasmas. Samples containing about 10 5 Sf-RVN or Sf9 cells were also analyzed for mycoplasmas using the Universal Mycoplasma Detection kit from the American Type Culture Collection (Manassas, VA), according to the manufacturer's protocol. This PCR-based assay uses primers complementary to sequences conserved in the 16S rRNA genes of more than 60 different species of mycoplasmas, acoleplasmas, spiroplasmas, and ureaplasmas, including eight species that are frequently found as cell culture contaminants. The results shown in FIG. 4 showed that neither Sf9 nor Sf-RVN cells were detectably contaminated with mycoplasmas. The absence of a PCR product was not due to inhibition of the PCR reaction by the insect cell lysates, as amplicons of the expected sizes were observed in PCRs performed using lysates spiked with the control templates (FIG. 4).

Example 7. Growth properties, morphologies and cell diameters. Sf-RVN or Sf9 cells were seeded at an initial density of 1.0 x 106 cells/mL in 50 mL shake flask cultures, triplicate samples were removed every 24 h for 4 days, and cell densities and sizes were measured. viable cells using a COUNTESS® automated cell counter (ThermoFisher Scientific, Inc.). Doubling times were calculated using the formula: Td = T x Log2/Log(Q2/Q1) where Td = doubling time, T = time (h) elapsed since the last passage, Q1 = cell seeding density and Q2 = viable cell count. Cell morphologies were documented by collecting phase contrast images at 10X magnification using an Olympus FSX-100 microscope and FSX-BSW imaging software (Olympus Life Sciences Solutions, Center Valley, Pennsylvania).

To compare several general properties of Sf-RVN cells with those of Sf9, their culture densities, diameters, doubling times, morphologies, and viabilities in response to baculovirus infection were evaluated. The results showed that Sf-RVN and Sf9 cells reached nearly identical mean densities within four days after being seeded in parallel shake flask cultures in ESF-921 medium (FIG. 5A). This time frame encompassed the 2-3 days of growth normally allowed between serial passages during routine maintenance of insect cell lines. The results also revealed no significant differences in the average diameters (FIG. 5B), doubling times (FIG. 5C), or morphologies (FIG. 5D) of Sf-RVN and Sf9 cells during the course of these cell culture experiments. . Finally, we found no significant differences in Sf-RVN and Sf9 cell viabilities in response to baculovirus infection, which were indistinguishable for 4 days post-infection at multiplicities of 0.1 (FIG. 6A) or 5 (FIG. 6B) cfu/cell. The time frame and two different MOIs used in this experiment encompassed the conditions typically used to produce working stocks of baculovirus or recombinant proteins at low or high MOIs, respectively. Overall, the results of these experiments demonstrated that the general properties of the Sf-RVN and Sf9 cells examined in this study are indistinguishable.

Example 8. Baculovirus expression vectors. A baculovirus expression vector designated BacPAK6-AChi/Cath encoding E. coli p-galactosidase (p-gal) was produced in two sequential steps. In the first step, BacPAK6 viral DNA was recombined with a plasmid encoding E. coli p-glucuronidase under the control of the baculovirus p6.9 promoter. In this plasmid, the p6.9-p-glucuronidase gene was inserted in place of the AcMNPV chiA and v-cath genes and embedded within AcMNPV wild-type flanking sequences. The desired recombinant was tentatively identified by its blue plaque phenotype in the presence of X-GlcA (RPI Corp., Mount Prospect, IL). The recombination site was confirmed by PCR with primers specific for the p-glucuronidase gene and 5'UTR of the AcMNPV gp64 gene, which were internal and external to the transfer plasmid, respectively. This virus was amplified and the viral DNA was isolated and digested with I-sceI to remove the entire p-glucuronidase expression cassette. In the second step, Sf9 cells were transfected with I-sceI digested viral DNA. The resulting progeny were resolved by plaque assay in the presence of X-GlcA and the final recombinant baculovirus, BacPAK6-AChi/Cath, was identified by its white plaque phenotype.

Recombinant baculovirus expression vectors designated AcP(-)p6.9hSEAP and AcP(-)p6.9hEPO encoded 8X HIS-tagged forms of human secreted alkaline phosphatase (hSEAP) and human erythropoietin (hEPO), respectively, under the control of AcMNPV p6.9 promoters and bee prepromellitin signal peptides. Synthetic genes encoding mature SEAP and EPO (Genbank NP_001623.3, amino acids 23-511 and Genbank NP_000790.2, amino acids 28-193, respectively) with N-terminal TEV protease cleavage sites (ENLYFQG) were designed using OPTIMIZER ( Puigbo et al., 2007) to match AcMNPV codon usage (http://www.kazusa.or.jp). These sequences were synthesized, cloned, and sequenced by Genscript (Piscataway, NJ), and error-free clones were used to produce recombinant baculovirus expression vectors by in vitro recombination with Ac6.9GT, as previously described (Toth et al. , 2011).

Standard methods were used to plaque purify, amplify and titrate recombinant baculovirus expression vectors in Sf9 cells. In addition, free stocks of Sf-rhabdovirus and Tnnodavirus were produced for this study. First, Sf9 cells were infected with working stocks of each baculovirus vector, and then baculoviral DNA was isolated using a standard method. This method includes treatments with proteinase K, SDS and RNaseA, followed by phenol/chloroform/isoamyl alcohol extraction and isopropanol precipitation of DNA, which was expected to remove Sf-rhabdoviruses and Tn-nodaviruses. The resulting baculoviral DNA preparations were then used to transfect Sf-RVN cells, and the progeny were plaque-purified, amplified, and titrated, except that Sf-RVN were used as hosts for plaque purification and amplification, rather than Sf9 cells. During this process, we analyzed extracts from baculoviral DNA-transfected and baculovirus-infected Sf-RVN cells, as well as pellets obtained by ultracentrifugation of samples from the final working virus stocks, for the presence or absence of Sf-rhabdovirus and Tnnodavirus using the RT-PCR/Nested PCR assays described in Example 4. No Sf-rhabdovirus or Tn-nodavirus sequences were detected.

Example 9. Expression of recombinant protein. Sf-RVN or Sf9 cells were seeded in ESF 921 culture media in six-well plates at densities of 1 x 10 6 cells/well. Cells were then mock-infected with ESF 921 medium or infected with Sf-rhabdovirus-free stocks of BacPAK6-AChi/Cath, AcP(-)p6.9hSEAP, or AcP(-)p6.9hEPO at multiplicities of infection. (MOI) of 0.1 or 5 plaque-forming units (pfu)/cell. At various time points post-infection, infected cells were harvested, cell densities were measured, and cells were pelleted by low speed centrifugation. Cells and cell-free media were then processed in various ways, depending on the nature of the model protein being expressed and the purpose of the experiment, as described below. In each case, however, recombinant protein levels in cell extracts and/or cell-free media were measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; (Laemmli, 1970)) and immunoblotting (Towbin et al. ., 1979) with protein- or tag-specific primary antibodies and alkaline phosphatase-conjugated secondary antibodies, as specified below. Immunoreactive proteins were visualized using a standard alkaline phosphatase-based color reaction and relative intensities were estimated by scanning and quantifying the bands using Image J software version 1.48 (US National Institutes of Health).

For p-gal, infected cell pellets were used to prepare cytoplasmic extracts for enzyme activity assays, using a known method. Western blotting was performed using rabbit anti-p-gal (EMD Millipore Corporation, Germany) and alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO) as primary and secondary probes, respectively.

For hSEAP, infected cell-free media were prepared for enzyme activity assays, and immunoblotting was performed using mouse anti-penta-His (ThermoFisher) and alkaline phosphatase-conjugated rabbit anti-mouse IgG (Sigma-Aldrich) as probes. primary and secondary, respectively.

For hEPO, infected cell-free media were prepared for immunoblotting with rabbit anti-hEPO (U-CyTech, Utrecht, The Netherlands) and alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma-Aldrich) as primary and secondary probes. , respectively.

The levels of baculovirus-mediated recombinant protein production supported by Sf-RVN and Sf9 cells were compared, using p-gal from E. coli , a model bacterial intracellular protein; hSEAP, a model human secreted glycoprotein; and hEPO, a model human secreted glycoprotein of biotechnological importance. It is important to emphasize that Sf-rhabdovirus-free working stocks of each of the recombinant baculoviruses were prepared and used for these studies, as described above.

E. coli p-gal expression experiments showed that there were no significant differences in the levels of intracellular enzyme activity produced by Sf-RVN and Sf9 cells during 4 days of infection with the recombinant baculovirus (FIG. 7A). Representative immunoblot results, shown in FIG. 7B, indicated that Sf-RVN produced slightly more total intracellular p-gal protein. An independent biological replicate in which the gel was stained with Coomassie Brilliant Blue produced the same result, but as in the results shown in FIGS. 7A-7C, the increase in intracellular p-gal levels produced by Sf-RVN cells was less (data not shown). Finally, it was observed that the levels of immunoreactive intracellular p-gal and enzyme activity produced by Sf-RVN and Sf9 cells were lower at 4 days post-infection, compared to earlier time points, which might reflect baculovirus-induced cytotoxicity at this very late time in infection.

Analysis of hSEAP production and secretion over 4 days of infection gave essentially the same results. In this case, we expanded the experiment to include low (0.1 pfu/cell; FIG. 8A, 8B, and 8C) and high (5 pfu/cell; FIG. 8D, 8E, and 8F) MOI infections because some researchers have reported of higher productivity with low MOI infections, rather than conventional high MOI infections in the BICS. The results of these experiments showed that there were no statistically significant differences in the activity levels of hSEAP produced by Sf-RVN and Sf9 cells infected with low (FIG. 8A) or high (FIG. 8D) MOIs. Representative immunoblot results indicated that Sf9 produced slightly more hSEAP when infected at low MOI (FIGS. 8B and 8C) and Sf-RVN produced slightly more hSEAP when infected at high MOI (FIGS. 8E and ). 8F). However, these were only minor differences, which were not fully reproducible in an independent biological replicate of this experiment (data not shown). Both Sf-RVN and Sf9 cells produced more hSEAP activity and immunoreactive extracellular protein at 1 day and approximately 3-fold more hSEAP activity at 4 days post-infection when infected at high MOI. It was also observed that there was no difference in the viabilities of Sf-RVN and Sf9 cells over 4 days of infection at either MOI, as shown in FIGS. 6A-6B, which was derived from data obtained as part of the hSEAP expression and secretion experiments described herein.

Finally, the same general results were obtained when comparing the levels of hEPO production and secretion by Sf-RVN and Sf9 cells. As there is no simple functional assay for this product, the analysis was limited to comparing the levels of immunoreactive hEPO secreted into the extracellular medium by the two different cell types during a 4-day infection. Results from two independent biological replicates of this experiment revealed no reproducible significant differences in the levels of secreted hEPO produced by Sf-RVN and Sf9 cells (Figures 9A and 9B).

Taken together, these results demonstrated that Sf-RVN and Sf9 cells produce and secrete three different recombinant proteins at nearly identical levels.

Example 10. Analysis of W-glycans. Fifty mL shake flask cultures of Sf-RVN and Sf9 cells were infected with Sf-rhabdovirus-free stocks of AcP(-)p6.9hEPO, and hEPO was affinity purified from cell- and virus-free supernatants using a Ni-NTA resin (ThermoFisher). W-glycans were enzymatically released from purified hEPO preparations by digestion with PNGase-F (New England Biolabs), and the released W-glycans were purified, derivatized and analyzed by MALDI-TOF-MS according to known methods. Structures were assigned to peaks based on predicted masses and knowledge of the W-glycans produced in Sf cells, annotated using standard bulleted symbols, and numbered for simplicity. Relative quantification of the different structures was achieved by dividing the combined maximum intensities of isotopic groups of individual permethylated W-glycan structures by the total intensity of all annotated W-glycan peaks.

Another important factor to assess when comparing Sf-RVN and Sf9 cells is their protein W-glycosylation patterns, as the patterns provided by the different cell lines can be drastically different. Therefore, Sf9 and Sf-RVN cells were infected with AcP(-)p6.9hEPO, secreted hEPO was purified from cell-free media, total W-glycans were enzymatically released, and permethylated products were analyzed by MALDI- TOF MS. The results showed that the vast majority of the hEPO-bound W-glycans produced by both cell lines had trimannosyl core structures (structures 2 and 3 of FIG. 10A), as expected. We also observed small proportions of hybrid-like structures with a terminal N-acetylglucosamine residue (structures 4 and 5 in FIG. 10A), as expected. By quantifying these different structures, we determined that the W -glycosylation profiles of hEPO provided by Sf-RVN and Sf9 cells were nearly identical, although the Sf-RVN cell product had slightly more fucosylated W-glycans (Figure 10B).

Example 11. Sf-RVN cells produce more infectious baculovirus progeny. In addition to their utility as hosts for the production of recombinant proteins, Sf9 cells are widely considered among the best hosts for the production of baculovirus stocks. Therefore, it was of interest to compare the amounts of infectious recombinant baculoviral vector progeny produced by Sf9 and Sf-RVN cells. This experiment involved infecting both cell types with two different Sf-rhabdovirus-free baculovirus pools, AcP(-)p6.9hEPO and AcP(-)p6.9hSEAP, collecting germinated viral progeny, i.e., cell culture media containing comprise infectious recombinant baculoviruses, from all four infections, and comparing infectious viral titers in plaque assays, as described in Example 8. Results from three independent biological replicates showed that the working pools of both baculoviruses, AcP(-) p6.9hEPO and AcP(-)p6.9hSEAP had approximately 5-10-fold higher titers when produced by Sf-RVN compared to those produced by Sf9 cells (FIG. 11).

Example 12. BLAST searches. Bioinformatic searches of the Sf cell genome and transcriptome were performed using the publicly accessible NCBI BLASTN interface (blast.ncbi.nlm.nih.gov/blast/Blast.cgi). The assembly of the transcribed sequence of the cell line Sf-21 (Genbank accession number GCTM00000000.1, BioProjectID 271593 (Kakumani et al., Biol. Direct 10, 1-7, 2015) and the assembly of the transcribed sequence of the fall armyworm Spodoptera frugiperda (Genbank accession number GESP00000000.1, BioProjectlD 318819 (Cinel et al.)) were queried against the published Sf-rhabdovirus genome (Genbank accession number NC_025382.1) using megaBLAST with default settings.

Results obtained from a megaBLAST search using the published Sf-rhabdovirus genome (Genbank accession number NC_025382.1) as a query against the transcribed sequence assembly of the cell line IPLB-SF-21 (Genbank accession number NC_025382.1). GCTM00000000.1, BioProjectlD 271593 (Kakumani et al., Biol. Direct 10, 1-7, 2015) are shown in Table 2.

Table 2

These results indicate that the Sf-21 cell line transcriptome includes the assembled and intact Sf-rhabdovirus genome. Since the Sf-21 cell line was previously shown to be persistently infected with Sf-rhabdovirus, this result was expected.

Results obtained from a megaBLAST search using the published Sf-rhabdovirus genome (Genbank accession number NC_025382.1) as a query against assembled whole-brain gene expression profiles of post-emergence adult male Spodoptera frugiperda (roundworm). fall armyworm), obtained from BioProject PRJNA318819 of the National Center for Biotechnological Information (Cinel et al.) are shown in Table 3.

Table 3

Surprisingly, several assembled sequences were detected in the transcriptome of these organisms, collectively comprising an intact Sf-rhabdovirus genome. These data show that the caterpillar ( Spodoptera frugiperda), from which all Sf cell lines are derived, is infected with Sf-rhabdovirus. Therefore, the reason why all cell lines derived from Spodoptera frugiperda are contaminated with Sf-rhabdovirus is that the organism was naturally infected with this virus in the environment before the first Sf cell line was isolated, not because the cell line(s) will be contaminated with the virus in the laboratory.

In sharp contrast, a BLAST search of the Sf-RVN transcriptome with the Sf-rhabdovirus sequence as query produced no matches, further confirming the finding that Sf-RVN cells are not contaminated with Sf-rhabdovirus. The results of the BLAST search are summarized in Table 4. Since all Sf cell lines previously analyzed by the laboratory and others were shown to be positive for Sf-rhabdovirus contamination, the absence of Sf-rhabdovirus sequences in the transcriptome of the Sf-RVN cell line clearly demonstrates that these cells are structurally (genetically) different from any other Sf cell line and from Spodoptera frugiperda, the natural organism from which all Sf cell lines described above are derived. This structural difference is supported by the fact that Sf-RVN cells are susceptible to infection by Sf-rhabdovirus.

Table 4. BLAST search results for Sf-rhabdovirus.

Example 13. The Sf-RVN cell line is susceptible to infection by Sf-rhabdovirus. Sf9 cells were seeded at an initial density of 1.0 x 106 cells per mL in TNM-FH medium supplemented with 10% (v/v) fetal bovine serum (Atlanta Biologicals, Inc., Flowery Branch, GA) in a culture in 50 ml shake flask. Cells were incubated at 28°C in a shaking incubator for 3 days. After incubation, cells were pelleted by low speed centrifugation and cell-free supernatants filtered through a 0.22 pM filter (CELLTREAT Scientific, Shirley, MA). This filtrate was used as Sf-rhabdovirus inoculum to examine the susceptibility of Sf-RVN cells to this virus.

For the infectivity experiment, duplicate Sf-RVN cells were seeded at a density of 2.0 x 106 cells in 5 mL of ESF921 medium (Expression Systems, Woodland, CA) in 25-cm2 flasks and incubated for 1 h at 28 h. °C to allow cells to adhere. Culture medium and cells in replicate flasks were then removed: (1) mock-infected with 2.5 mL TNM-FH medium supplemented with 10% (v/v) fetal bovine serum or (2) ) were infected with 2.5 ml of the Sf-rhabdovirus inoculum described above. Cells were incubated for 2 h at 28 °C and then 2.5 mL fresh TNM-FH medium supplemented with 10% (v/v) fetal bovine serum was added and cells were incubated for another 24 h at 28 °C. At 24 h after infection, a pool of infected cells was washed three times with mock rhabdovirus inoculum or Sf and harvested. The second set was further incubated at 28 °C, sampled and serially passaged (P0 to P1) 72 h post-infection, and then sampled and serially passaged again after two additional incubation periods. 72 h until a total of three passages were performed. Samples obtained at each passage level were used to produce cell pellets by low-speed centrifugation, total RNA was extracted, and samples were analyzed by RT-PCR, as described in Example 4.

No Sf-rhabdovirus-specific amplicon was observed with RNA obtained from mock-infected Sf-RVN cells at any time point or with RNA obtained from P0 Sf-RVN cells infected with Sf-rhabdovirus inoculum at 24 or 72 hours. h after infection (FIGS. 13A-13B). However, a faint amplicon was observed with the RNA obtained from Sf-RVN cells infected with the Sf-rhabdovirus inoculum at 72 h after P1 and its intensity increased progressively with the RNA obtained at 72 h after P2 and at 72 h after P3 (FIG. 13A-13B). These results clearly demonstrate that Sf-RVN cells are susceptible to infection with the Sf-rhabdovirus produced by the contaminated Sf cells.

Example 14. Conventional methods fail to produce a virus-free established T. ni cell line. We also attempted to isolate Tn-nodavirus-free cells by culturing polyclonal TN-368 cell populations in TNM-FH medium supplemented with 10% (v/v) fetal bovine serum plus various concentrations of a cocktail of antiviral drugs including ribavirin, 6-azauridine and vidarabine. Cells were cultured with these three drugs for 15 days with custom serial passages, and samples were routinely analyzed for Tn-nodavirus by RT-PCR, as described in Example 16. As shown in Figs. FIGS. 13A-13B, amplicons corresponding to Tn-nodavirus segments 1 and 2 were present in TN-368 cells that had been incubated at all concentrations of the tested antiviral cocktail (FIGS. 13A and 13B, respectively). Therefore, as with Sf9 cells, neither nodavirus-free nor T cells could be obtained using populations of TN-368 cells treated with this antiviral cocktail.

Example 15. Exemplary method for obtaining an established T. ni cell line lacking virus. After discovering that polyclonal TN-368 cell cultures treated with antiviral drug cocktails remained positive for Tn-nodavirus, a described method was used to obtain a virus-free cell line. This exemplary method embodiment comprised isolating single TN-368 cells by limiting dilution to isolate single cells, seeding the isolated cells in 96-well plates in TNM-FH medium supplemented with 10% (v/v) fetal bovine serum and 200 pg/ml of ribavirin to form a first culture composition. The first culture composition was grown for approximately one month with custom amplification to produce progressively larger cultures, and after reaching the 25 cm2 flask level, samples were tested for Tn-nodavirus by RT-PCR, followed by Nested PCR, as described in Example 16. A clone lacking Tn-nodavirus (FIG. 14A, lane CL#3) was transferred to media lacking antiviral drugs to form a second culture composition. The clone, designated passage zero (P0) of Tn-NVN, was adapted to ESF 921 medium without serum and cultured in suspension. The Tn-NVN cell line was subsequently maintained in this second culture composition and growth format.

Example 16. Tn-nodavirus-specific reverse transcription PCR (RT-PCR)/Nested PCR. Samples of TN-368 and Tn-NVN cultures containing 1 x 10 6 cells were collected, and the cells pelleted by low speed centrifugation. Cell-free supernatants were filtered through a 0.22 pm filter (CELLTREAT Scientific, Shirley, MA) and then ultracentrifuged at 131,000 xg for 22 h at 4°C. Total RNA was extracted from both low-speed cell pellets and high-speed cell-free pellets using RNASo/y reagent (Omega Bio-Tek, Inc., Norcross, GA), according to the manufacturer's protocol. RNAs were then quantified and used as templates for cDNA synthesis with the ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA) and a Tn-nodavirus-specific primer designated Noda-7 (SEQ ID N°: 24), according to the manufacturer's protocol. Equivalent amounts of each cDNA preparation were used for nested PCRs with Taq DNA polymerase, ThermoPol reaction buffer (New England Biolabs), and segment-specific primer pairs 1 (Noda-1; SEQ ID NO: 19 and Noda-2). ; SEQ ID NO: 20) or segment 2 (Noda-6; SEQ ID NO: 23 and Noda-7; SEQ ID NO: 24) of Tn-nodavirus RNA. The reaction mixtures were incubated at 94 °C for 3 min, cycled 35 times at 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 1 min, and finally incubated at 72 °C for 10 s. min. One pL of each primary PCR was then used as a template for nested PCRs under the same conditions with segment 1-specific primer pairs (Noda-1i; SEQ ID NO: 21 and Noda-2i; SEQ ID NO: : 22) or segment 2 (Noda-6i; SEQ ID NO: 25 and Noda-7i; SEQ ID NO: 26) of Tn-nodavirus RNA. The products of RT-PCR and RT-PCR followed by nested PCR were analyzed by ethidium bromide-stained agarose gel electrophoresis according to standard methodology. The sequence of each primer used for these assays is shown in Table 5.

Example 17. n-NVN T cells have no detectable n-nodavirus T. Total T n-NVN RNA was isolated at various passage levels and analyzed for the presence of Tn-nodavirus by RT-PCR, followed by nested PCR with primers specific for segment 1 (FIG. 15A) or 2 (FIG. 15A). 15B) of Tn-nodavirus, as described in Example 16. The results of Tn-nodavirus-specific nested RT-PCR/PCR demonstrated that Tn-NVN cells had no detectable Tn-nodavirus for at least 55 serial passages. in the absence of any antiviral drug (FIGS. 15A-15C). Total RNA was also isolated from the pellet fraction obtained by ultracentrifugation of cell-free media (CFM) from Tn-NVN cells at passage 55 and used to analyze Tn-nodavirus using specific nested RT-PCR/PCR. of Tn-nodavirus, as described in Example 16. As shown in FIG. 15C, a Tn-nodavirus amplicon was observed in the lanes corresponding to RNA isolated from TN-368 cells and from the TN-368 cell-free medium pellet. In contrast, the Tn-nodavirus amplicon was not detected in the RNA isolated from the Tn-NVN cell-free media pellet. All results shown in FIGS. 15A-15C were obtained using RNA from cells cultured in the absence of antiviral drugs. Together, these results demonstrated that there was no detectable Tn-nodavirus RNA in Tn-NVN cells or cell-free media over the course of 55 passages in the absence of antiviral drugs, indicating that these cells are Tn-nodavirus-free.

Example 18. Detection of mycoplasmas. Samples containing about 10 5 Tn-NVN or TN-368 cells were also analyzed for mycoplasmas using the ATCC (Manassas, VA) Universal Mycoplasma Detection kit, according to the manufacturer's protocol. This PCR-based assay uses primers complementary to sequences conserved in the 16S rRNA genes of more than 60 different species of mycoplasmas, acoleplasmas, spiroplasmas, and ureaplasmas, including eight species that are frequently found as cell culture contaminants. The results shown in FIG. 16 showed that neither TN-368 nor Tn-NVN cells were detectably contaminated with mycoplasmas. In both cases, the absence of a PCR product was not due to inhibition of the PCR reaction by the insect cell lysates, since amplicons of the expected sizes were observed in PCRs performed using lysates enriched with the control templates (FIG. 16, lanes Tn-NVN (+) and TN-368 (+)).

Example 19. Cell growth properties, morphology and diameter of Tn-NVN and TN-368 cells. The general properties of Tn-NVN cells were compared with those of TN-368, including their culture densities, diameters, and morphologies using the techniques described in Example 7. The results showed that Tn-NVN and TN-368 cells achieved virtually identical average densities within five days after being seeded in parallel shake flask cultures in ESF-921 medium (FIG. 17A). The results also revealed no significant differences in the average diameters (FIG. 17B) or morphologies (FIG. 17C) of Tn-NVN and TN-368 cells during the course of these cell culture experiments. In general, these results demonstrated that the general properties of the Tn-NVN and TN-368 cells examined in this study are the same or substantially the same.

Example 20. Tn-NVN and TN-368 cells produce recombinant proteins at almost identical levels. We also compared the levels of baculovirus-mediated recombinant protein production maintained by Tn-NVN and TN-368 cells, using p-gal, hSEAP, and hEPO, as described in Example 9. It is important to emphasize that free working stocks were prepared. of Tn-nodavirus from each of the recombinant baculoviruses and used for these studies, as described in Example 8.

p-gal expression experiments in E. coli revealed no significant differences in the activity levels of the intracellular enzyme (FIG. 18A) or total intracellular p-gal protein (FIGS. 18B and 18C) produced by Tn cells. -NVN and TN-368 over the course of 4 days of infection. Again, we noted that immunoreactive intracellular p-gal and enzyme activity levels decreased at 3–4 days post-infection, compared to earlier time points, perhaps reflecting baculovirus-induced cytotoxicity at these later times. of infection.

Analysis of hSEAP production and secretion over the course of 4 days of infection yielded essentially the same results, revealing no statistically significant differences in the activity levels of hSEAP or secreted immunoreactive hSEAP protein produced by Tn-NVN and TN cells. -368 (FIGS. 19A-19C).

Finally, we obtained the same general results when we compared the levels of hEPO production and secretion by Tn-NVN and TN-368 cells during a 4-day infection (Figures 20A-20B). These results demonstrated that Tn-NVN and TN-368 cells produce and secrete three different recombinant proteins at the same or substantially the same level.

Example 21. W-glycan analysis of n-NVN and TN-368 T cells. The W-glycosylation profiles of Tn-NVN and TN-368 cells were analyzed, as described in Example 10. MALDI-TOF-MS analysis of the isolated W-glycans of hEPO produced by Tn-NVN and TN- 368 showed that they provided essentially identical glycosylation patterns. The vast majority of W-glycans in hEPO from both cell lines were structures with a bimannosyl core. FIG. 21A, structure 1), but small proportions of structures with fucosylated trimannosyl cores with and without terminal W-acetylglucosamine residues were also observed (Figure 21A, structures 3 and 6).

Example 22. The BmN cell line, derived from Bombyx mori, is infected with Sf-rhabdovirus. To investigate whether the cells of the lepidopteran insect Bombyx Mori are contaminated, we tested the BmN cell line for the presence of Sf-rhabdovirus. A vial of BmN cells (ATCC-CRL 8910) was thawed from the laboratory cell bank, the cells were pelleted by low-speed centrifugation, and total RNAs were extracted, quantified, and analyzed by RT-PCR, as described in Example 4. RT-PCRs were performed essentially as described, except that in this case independent RT-PCRs were performed with primers specific for all six Sf-rhabdovirus genes (Table 6).

As shown in Fig. 22, amplicons corresponding to the six Sf-rhabdovirus genes (N, P, M, G, X and L) were observed. These results demonstrated that the BmN cell line, which is derived from the lepidopteran insect Bombyx mori, a close relative of S. Frugiperda, is also infected with Sf-rhabdovirus.

Taken together, the results suggest that many established cell lines may be infected with a virus. This may be due to persistent viral infection of the organisms from which these cell lines are derived. Our results also demonstrate that the reported methods for obtaining established lines are widely applicable.

The recent flurry of regulatory agency approvals for the use of BICS-derived biologics in human and veterinary patients is a critical milestone in the emergence of BICS as a true commercial biologics manufacturing platform. However, the discovery of infectious viral contaminants in insect cell lines most commonly used as hosts for baculovirus vectors, including Sf and Tn cells, raises questions about the safety of biologics produced by BICS. In this context, it is important to emphasize that there is no evidence that Sf-rhabdoviruses or Tnnodaviruses pose a clear threat to human or veterinary patients. However, the clear answer to identifying any adventitious agent in any bio-manufacturing platform is to remove the agent to create an inherently safer system. Therefore, Sf-RVN and Tn-NVN, which are not contaminated with Sf-rhabdovirus or Tn-nodavirus, respectively, have been invented. In fact, both cell lines lack any detectable trace of either of these newly identified viral contaminants.

This conclusion is based on the results of highly sensitive RT-PCR/Nested PCR assays, which were used to demonstrate that Sf-RVN cells and cell-free media had no detectable Sf-rhabdovirus RNA, and Tn-NVN cells and cell-free media had no detectable Tn-nodavirus RNA for at least 55 passages in the laboratory. It is strongly supported by the length of the Sf-rhabdovirus and T n-nodavirus-specific RT-PCR/Nested PCR testing regimens, which are still ongoing and, at this time, have revealed no traces of Sf-rhabdovirus in Sf -RVN or Tn-nodavirus in Tn-NVN cells over the course of 170 and 100 serial passages, respectively. If these cells had a low level of Sf-rhabdoviral or Tn-nodaviral contamination, these viruses would be expected to replicate quite rapidly to detectable levels, particularly considering the high level of contamination reported (2 X 109 particles/mL of extracellular growth medium). ) in Sf cell cultures. Furthermore, the Sf-RVN results have been confirmed and extended by bioinformatic analyzes of publicly available genomic and transcriptomic data on Sf-21 cells (Geisler and Jarvis, 2016), as well as original genomic and transcriptomic databases obtained by massively parallel sequencing. of Sf-RVN cells (Table 2).

Another conclusion of current teachings is that the essential properties of Sf-RVN and Tn-NVN cells, in the context of their potential as alternative hosts for BICS, are very similar to those of Sf9 and TN-368 cells, respectively. which were used as "reference" hosts for BICS due to their widespread use in this field. Neither Sf-RVN cells nor Tn-NVN cells were found to be detectably contaminated with mycoplasmas. The basic growth properties of Sf-RVN and Sf9 and Tn-NVN and TN-368 cells, respectively, examined within the parameters of standard cell culture maintenance protocols, were also found to be indistinguishable.

Another conclusion of current teachings is that Sf-RVN and Tn-NVN cells can function at least as well as their virus-contaminated counterparts as host components of BICS. This conclusion was supported by the finding that Sf-RVN and Sf9 and Tn-NVN and TN-368 cells, respectively, supported approximately equal levels of recombinant protein and glycoprotein production, secretion, and enzymatic activity. Formally, this conclusion can only be applied to the three different products used as models in this specification. Although one intracellular bacterial protein and two secreted human W-glycoproteins were used in an effort to broaden the analysis, it is possible that Sf-RVN and/or Tn-NVN cells will produce higher or lower levels of other recombinant proteins in the future. . It was also found that Sf-RVN and Sf9 and T n-NVN and TN-368 cells, respectively, gave nearly identical W-glycosylation patterns. The conclusion that Sf-RVN and Sf9 and Tn-NVN and TN-368 cells gave almost identical W-glycosylation patterns formally applies only to hEPO, which was the model used for the analysis. However, compared to the potential variation in recombinant protein production levels, it is much less likely that Sf-RVN and Sf9 and Tn-NVN and TN-368 W-glycosylate differentially other products, because the analytical results obtained with a given product reflect endogenous W-glycan processing capabilities. If they existed, the differences in the degree of processing of W-glycans would have been detected in the analysis of glycosylation of hEPO by the different cell lines.

Another important functional ability of the Sf-RVN and Sf9 cells examined herein was their ability to produce infectious recombinant baculovirus progeny. Surprisingly, Sf-RVN cells were found to produce higher levels of infectious progeny (in some cases five to ten times more) when used to propagate two different recombinant baculoviruses, compared to Sf9 cells (FIG. 11). This difference was statistically significant and demonstrates a clear advantage of Sf-RVN cells over Sf9 cells.

Although the disclosed teachings have been described with reference to various applications, methods, and compositions, it will be appreciated that various changes and modifications may be made without departing from the teachings herein. The above examples are provided to better illustrate the present teachings and are not intended to limit the scope of the teachings herein. In addition, those skilled in the art can perform

Claims (5)

1. A method for obtaining a Spodoptera frugiperda cell line free of Sf-rhabdovirus, comprising: isolating a cell from a Spodoptera frugiperda organism contaminated with Sf-rhabdovirus or from a Spodoptera frugiperda cell line contaminated with Sf-rhabdovirus; combining the isolated cell with a cell culture medium comprising an antiviral compound to form a first culture composition; incubating the first culture composition under conditions suitable for the cell to grow and divide, thereby generating a multiplicity of cells; removing a portion of the multiplicity of cells or cell culture medium and testing for the presence or absence of a virus; combining at least part of the multiplicity of cells with cell culture media without an antiviral compound to form a second culture composition; Y incubating the second culture composition under suitable conditions for the cells to grow and divide, thus obtaining a virus-free cell line. 2. The method of claim 1, wherein the virus contaminated cell line comprises Sf-21 cells or Sf9 cells. 3. The method of claim 1, wherein the analysis comprises (a) RT-PCR, (b) RT-PCR and nested PCR; (c) Quantitative RT-PCR; or (d) an antibody-based detection technique. 4. The method of claim 1, wherein the antiviral compound is a nucleoside analog, optionally wherein the nucleoside analog comprises at least one of: ribavirin, 6-azauridine, vidarabine, acyclovir, 9-/3- D-arabinofuranosyladenine (Ara-A), cytosine arabinose, adenine arabinoside, and guanine 7-N-oxide (G-7-Ox), optionally where the nucleoside analog is 6-azauridine. 5. The method of claim 1, wherein the isolation comprises limiting dilution; where the antiviral compound is 6-azauridine; and wherein the analysis comprises: (a) RT-PCR or (b) RT-PCR and nested PCR.